We have completed a study of the DNA binding properties of a ubiquitous nuclear protein isolated in our laboratory as a transcription factor (EBP-80) for mouse intracisternal A-particle LTRs and subsequently found to be the same as a previously known protein (Ku) first detected by antibodies from certain autoimmune patients. Ku is known to bind to the ends of duplex DNA. Our study confirms that EBP/Ku binds with high affinity (Kd's of 10-40 pM) to blunt-ended duplex DNA. However, it binds equally strongly to duplex DNA in which the strands are connected at either end by hairpin loops (i.e., there are no free strand ends), and to constructs consisting of short stretches of duplex DNA with one or more single strand extensions. EBP/Ku also binds to circular duplex molecules containing a short single-stranded region or a double-stranded region of non-homology (bubble), but does not bind to the corresponding construct consisting entirely of duplex DNA. We conclude that EBP/Ku recognizes single-to- double strand transitions in DNA. Binding to regions of strand separation could be related to the transcriptional enhancement observed by us and others, as well as to a possible role in DNA replication. The functional role of this protein is currently under study. We earlier identified a discrete and highly related subpopulation of IAP elements (LS elements) that are transcriptionally active in normal mouse B lymphocytes and thymus cells. Oligonucleotide probes specific for the three LS subfamilies each hybridize with a restricted number of fragments in gel-fractionated genomic restriction digests. We have found that each probe detects multiple polymorphisms between different strains of inbred mice and thus can be used for chromosome mapping of IAP elements. The LS probes are already in active use in this and other laboratories as multilocus probes for genomic mapping, linkage analysis and strain identification in inbred mice